Midkine (hereinafter abbreviated as “MK” as required) is a growth/differentiation factor that was first discovered as a gene product expressed transiently in the process of differentiation induction of embyonic tumor cells (EC) with retinoic acid, being a polypeptide having a molecular weight of 13 kDa, rich in basic amino acids and cysteine (see, for example, non-patent document 1 and non-patent document 2).
The steric structure of MK has been determined by NMR and reported (see, for example, non-patent document 3). When characterized structurally, MK is configured mainly with two domains. Specifically, MK consists of a fragment on the N-terminal side consisting of amino acid residues 1 to 52 (hereinafter referred to as “the N-terminal fragment”), a fragment on the C-terminal side consisting of amino acid residues 62 to 121 (hereinafter referred to as “the C-terminal fragment”) and a loop region that connects the fragments (amino acid residues 53 to 61). Bound to the outside of each domain is a tail that is rich in basic amino acids. In the MK molecule, each of the N-terminal fragment and the C-terminal fragment has a steric structure consisting mainly of three reversed β sheet structures (hereinafter referred to as “domains”; a domain consisting of the amino acid residues 15 to 52 in the N-terminal fragment referred to as “the N-domain”, a domain consisting of the amino acid residue 62 to 104 in the C-terminal fragment referred to as “the C-domain”), and freely moving structures assuming no particular structure (hereinafter referred to as “tails”; a tail consisting of the amino acid resideues 1 to 14 in the N-terminal fragment referred to as “the N-tail”, and a tail consisting of the amino acid resigues 105-121 in the C-terminal fragment referred to as “the C-tail”).
Known receptors of MK include receptor-type protein tyrosine phosphatase ζ (PTPζ), LRP (low density lipoprotein receptor-related protein), ALK (anaplastic leukemia kinase), integrin and syndecan and the like. MK is a highly positively charged protein containing large amounts of the basic amino acids lysine (K) and arginine (R). It has a heparin-binding site in the C-domain thereof, and is known to bind strongly to negatively charged molecules such as heparin and chondroitin sulfate E. As a result of mutagenesis analysis and NMR analysis, it is thought that cluster I, configured with K79, R81, and K102, and cluster II, configured with K86, K87, and R89, are important to the binding with heparin. Meanwhile, a report that only cluster I is important to the binding with chondroitin sulfate E is available. When R81 of cluster I is replaced with A, the binding activity with heparin decreases. As a result, the reduction of the binding activity to PTPζ and the MK-induced neurite elongation and movement of nerve cells are suppressed.
Some growth factors such as fibroblast growth factor (bFGF) and vascular endothelial cell growth factor (VEGF) have a heparin-binding site. These growth factors are thought to bind to heparan sulfate proteoglycan, an extracellular matrix, stay at appropriate positions, and are released as required. The same are also known to bind to heparan sulfate expressed in nerve cells and vascular endothelial cells to contribute to neurite elongation and fibrinolytic activity elevation. When a Petri dish is coated with MK and mouse embryo nerve cells are sown thereon, neurites elongate. In this situation, digestion of the nerve cells with heparitinase suppresses the neurite elongation. Meanwhile, when vascular endothelial cells are cultured and MK is added, the plasminogen Activator activity of the cells rises. In this case as well, digestion of the cells with heparitinase suppresses the elevation of plasminogen activity.
MK is thought to be bound with PTPζ at two sites. One site involves a high affinity bond with chondroitin sulfate (Kd=0.58 nM). This bond disappears upon digestion with chondroitinase. The other site involves a bond with protein, being a low-affinity bond that remains after digestion with chondroitinase (Kd=3 nM). MK promotes the migration of fetal nerve cells expressing PTPζ; treatment of the nerve cells with chondroitinase ABC suppresses the migration. Osteoblast-like UMR106 cells are expressing PTPζ, and are known to have the MK-dependent migration thereof suppressed by treatment with chondroitinase ABC. The MK-dependent migration of macrophage is also suppressed by treatment with chondroitinase ABC, chondroitinase B, or heparinase. Because macrophage is not thought to express PTPζ, is it thought that another receptor is involved.
Whatever negatively charged does not bind to the heparin-binding site of MK. When. MK was immobilized by aminocoupling and subjected to surface plasmon resonance analysis, the results obtained showed that chondroitin sulfate E and heparin bound strongly to MK, whereas chondroitin sulfate A, B, C, and D did not bind thereto.
MK is known to possess a broad range of biological activities. For example, it is known that in human cancer cells, the expression of MK is increased. This increased expression has been observed in a wide variety of cancers, including esophageal cancer, thyroid cancer, urinary bladder cancer, colorectal cancer, gastric cancer, pancreatic cancer, chest cancer, liver cancer, lung cancer, breast cancer, neuroblastoma, glioblastoma, uterine cancer, ovarian cancer, and Wilms' tumor (see, for example, patent document 1 and non-patent document 4). MK is also thought to promote the survival and movement of cancer cells and facilitate neovascularization to help the advancement of cancer.
MK is also known to be one of the molecules that play the central role in the process of development of inflammation. For example, it is known that the formation of nascent intima after blood vessel damage and the onset of nephritis in ischemic injury are mitigated in knockout mice lacking the MK gene. It is also known that in a rheumatism model, postoperative adhesion is also considerably mitigated in MK knockout mice (see, for example, patent document 2, patent document 3 and patent document 4). Hence, MK is known to be involved in inflammatory diseases such as arthritis, autoimmune disease, rheumatic arthritis (rheumatoid arthritis (RA), osteoarthritis (OA)), multiple sclerosis, postoperative adhesion, inflammatory colitis, psoriasis, lupus, asthma, and neutrophil functional abnormalities. Furthermore, MK is known to promote the movement (migration) of inflammatory cells such as macrophage and neutrophils. Because this movement is required for the development of inflammation, it is thought that when midkine is lacked, inflammation-based diseases are unlikely to occur. (See, for example, patent document 5).
Since MK levels are increased in the peritoneal fluid of females with advanced endometriosis, and also since MK stimulates the proliferation of cultured endometrial interstitial cells, MK is known to be involved in the onset and progression of endometriosis (see, for example, patent document 6).
Furthermore, exhibiting vascular intimal thickening action, MK is known to be involved in vascular obstructive diseases such as restenosis following vascular reconstruction surgery, cardiac coronary arterial vascular obstructive disease, cerebral vascular obstructive disease, renal vascular obstructive disease, peripheral vascular obstructive disease, arteriosclerosis, and cerebral infarction (see, for example, patent document 2).
Cell migration is known to be important to the mechanisms for cancer cell infiltration/metastasis, intimal thickening in arteriosclerotic foci, neovascularization and the like. It is also known that inflammatory cell migration is profoundly associated with cardiovascular diseases such as angina pectoris, myocardial infarction, cerebral infarction, cerebral hemorrhage, and hypertension.
Pleiotrophin (PTN or HB-GAM) is the only family protein of the MK, having approximately 50% homology to MK. Both MK and PTN are proteins containing large amounts of cysteine and basic residues. All the 10 cysteine residues are conserved in MK and PTN, and structurally, both can be divided into the N-domain and the C-domain. As a result of NMR analysis, it is known that these two molecules have very similar three-dimensional structures. Each domain consists of three β sheets, connected via a flexible linker region. K79, R81, and K102, considered to be important to the binding with chondroitin sulfate and heparin, are conserved between the two proteins. K79 and R81 are present on the same β sheet, whereas K102 is present on another β sheet. When MK and PTN form a steric structure, these basic residues appear in the vicinity of the protein surface.
In recent years, applications of RNA aptamers to therapeutic drugs, diagnostic reagents, and test reagents have been drawing attention; some RNA aptamers have already been in clinical stage or in practical stage. In December 2004, the world's first RNA aptamer drug, Macugen, was approved as a therapeutic drug for age-related macular degeneration in the US. An RNA aptamer refers to an RNA that binds specifically to a target substance such as a protein, and can be prepared using the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method (non-patent documents 5, 6). The SELEX method is a method by which an RNA that binds specifically to a target substance is selected from about 1014 RNA pools having different nucleotide sequences. The RNA used has a structure wherein a random sequence of about 40 residues is sandwiched by primer sequences. This RNA pools are allowed to associate with a target substance, and only the RNA that has bound to the target substance is recovered using a filter and the like. The RNA recovered is amplified by RT-PCR, and this is used as the template for the next round. By repeating this operation about 10 times, an RNA aptamer that binds specifically to the target substance can be sometimes acquired.    [patent document 1] JP-A-6-172218    [patent document 2] WO2000/10608    [patent document 3] WO2004/078210    [patent document 4] WO2004/085642    [patent document 5] WO1999/03493    [patent document 6] WO2006/016571    [non-patent document 1] Kadomatsu, K. et al., Biochem. Biophys. Res. Commun., 151:p. 1312-1318    [non-patent document 2] Tomokura, M. et al., J. Biol. Chem, 265: p. 10765-10770    [non-patent document 3] Iwasaki, W. et al., (1997) EMBO J. 16, p. 6936-6946    [non-patent document 4] Muramatsu, T., (2002) J. Biochem. 132, p. 359-371    [non-patent document 5] Ellington et al., (1990) Nature, 346, 818-822    [non-patent document 6] Tuerk et al., (1990) Science, 249, 505-510